Artificial plant promoter activated by broad spectrum of xanthomonas

ABSTRACT

The present invention relates to synthetic promoter and a synthetic gene which confers broad-spectrum disease resistance to Xanthomonands in plants. The present invention also relates to transgenic plants containing the synthetic gene and plants derived by crossing plants with such transgenic plants. More specifically, the synthetic promoter is a synthetic Xa10 promoter and the synthetic gene is a synthetic Xa10 gene which contains the synthetic Xa10 promoter. The resistance is resistance to bacterial blight and the plants are rice plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a national stage filing under 35 U.S.C. §371of PCT/SG2012/000414, filed on 1 Nov. 2012, which is incorporated hereinby reference in its entirety.

SEQUENCE SUBMISSION

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is entitled2577227SequenceListing.txt, created on 24 Oct. 2012 and is 29 kb insize. The information in the electronic format of the Sequence Listingis incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to synthetic promoter and a synthetic genewhich confers broad-spectrum disease resistance to Xanthomonands inplants. The present invention also relates to transgenic plantscontaining the synthetic gene and plants derived by crossing plants withsuch transgenic plants. More specifically, the synthetic promoter is asynthetic Xa10 promoter and the synthetic gene is a synthetic Xa10 genewhich contains the synthetic Xa10 promoter. The resistance is resistanceto bacterial blight and the plants are rice plants.

The publications and other materials used herein to illuminate thebackground of the invention or provide additional details respecting thepractice, are incorporated by reference, and for convenience arerespectively grouped in the Bibliography.

Gram-negative phytopathogenic bacteria employ a type III secretionsystem (TTSS) to translocate effector proteins into plant cells wherethey modulate host cell functions for the benefit of the invasionprocess (Alfano and Collmer, 2004; Kay and Bonas, 2009). Members of thelarge AvrBs3 effector family are unique kind of type III effectorsproduced by pathovars of Xanthomonas and Ralstonia solanacearum (Bonaset al., 1989; Yang and White, 2004; Heuer et al., 2007). AvrBs3-likeeffectors, also recently referred to as transcription activator-like(TAL) effectors (Yang et al., 2006; Bogdanove et al., 2010), have incommon an N-terminus required for type III secretion and a C-terminuscontaining nuclear localization signals (NLS) and an acidic activationdomain (AAD). TAL effectors differ in the middle, a region of typically33-35 amino acid (aa) long, near-perfect repeats that ends in a 20 aalong truncated repeat. The two hypervariable amino acids at position 12and 13 of repeats, also termed as repeat-variable di-residue (RVD)(Moscow and Bogdanove, 2009), contribute to repeat polymorphism, whereasthe number and order of repeats with polymorphic RVDs in a TAL effectordetermine the specific activity (Herbers et al., 1992; Yang et al.,2005).

Individual TAL effectors activate transcription of specific hostsusceptibility (S) genes for promoting disease development (Yang et al.,2006; Kay et al., 2007; Sugio et al., 2007; Antony et al., 2010). Inorder to counteract the disease-promoting strategies, plants haveevolved mechanisms that exploit the transcription inducing ability ofthe TAL effectors (Gu et al., 2005; Romer et al., 2007). Thus, a subsetof TAL effectors functions as avirulence effectors and activates thetranscription of disease R genes. TAL effectors bind specific DNAsequences in the promoters of S or R genes (Kay et al., 2007; Romer etal., 2007; Romer et al., 2009; Antony et al., 2010). Each RVD from thecentral repeats of TAL effectors specifically recognizes a nucleotide inthe target DNA element with a conserved T at the 5′ end (Boch and Bonas,2010; Bogdanove et al., 2010).

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight ofrice (Nino-Liu et al., 2006). Individual strains of X. oryzae pv. oryzaeharbor 11 to 19 TAL effectors (White et al., 2009). TAL effectors fromX. oryzae pv. oryzae target to rice genes for either susceptibility(Yang et al., 2006; Sugio et al., 2007; Chen et al., 2010) or resistanceto bacterial infection (Gu et al., 2005). TAL effector PthXo1 from X.oryzae pv. oryzae strain PXO99^(A) targets Os8N3/Xa13/OsSWEET11 in rice(Yang et al., 2006; Chen et al., 2010). The recessive allele of Xa13(xa13) is unresponsive to PthXo1, and plants with xa13 are resistant tostrains of the pathogen that rely solely on the PthXo1 as the essentialeffector for virulence (Yang et al., 2006). The xa13-mediated resistanceto rice bacterial blight can be defeated by induction of the S geneOs-11N3, another member of the N3 gene family, by strains of thepathogen utilizing the TAL effectors AvrXa7 and PthXo3 (Antony et al.,2010).

PthXo6 and PthXo7 are two other TAL effectors from PXO99^(A) and targetrespectively to two transcription factor genes, OsTFX1 and OsTFIIA γ1,in rice (Sugio et al., 2007). OsTFX1 encodes a bZIP transcription factorwhereas the gene product of OsTFIIA γ1 is the small subunit of thetranscription factor HA (Sugio et al., 2007). The induction of OsTFIIAγ1 located on chromosome 1 by PthXo7 may reflect the adaptation ofPXO99^(A) to the resistance mediated by xa5 (Iyer and McCouch, 2004), anallele of OsTFIIA γ5 encoding a second form of the TFIIA small subuniton chromosome 5 of rice (Sugio et al., 2007). The DNA target sequencesof PthXo1, EBE_(PthXo1) (Effector Binding Element for PthXo1), has beenidentified in the promoter of Os8N3 (Antony et al., 2010), whereas theDNA target sequences of PthXo6 and PthXo7 remain to be identified orverified in the promoters of OsTFX1 and OsTFIIA γ1, respectively,although the putative target sequences were predicted (Boch et al.,2009).

Three TAL effectors, AvrXa7 and AvrXal10 from PXO86 (Hopkins et al.,1992) and AvrXa27 from PXO99^(A) (Gu et al., 2005), activate diseaseresistance when rice plants carried the cognate R genes Xa7, Xa10 orXa27. So far, only the Xa27 gene has been isolated and published (Gu etal., 2005). Xa27 is induced by AvrXa27 and the gene can providenon-specific resistance to X. oryzae pv. oryzae if the AvrXa27-induciblepromoter is replaced with a stress-inducible promoter from the rice PR1gene (Gu et al., 2005; Tian and Yin, 2009). The full induction of Xa27by AvrXa27 requires OsTFIIA γ5, the gene product of Xa5 on chromosome 5(Gu et al., 2009). A 16- to 18-bp DNA cis-element, designated asUPT_(AvrXa27) (UPregulated by TAL effector AvrXa27 or EBE_(AvrXa27)),was identified in the promoter of Xa27 to be specifically induced byAvrXa27 (Boch et al., 2009; Romer et al., 2009).

Xa10 confers narrow-spectrum race-specific resistance to a fewPhilippine races of X. oryzae pv. oryzae (Yoshimura et al., 1995). The Rgene was introgressed from rice cultivar Cas 209 into susceptible ricevariety IR24 (Mew, 1982; Yoshimura et al., 1983). Xa10 was finely mappedto a genetic region of 0.28 cM between proximal marker M491 and distalmarker M419 on the long arm of chromosome 11 and co-segregated withmarkers S723 and M604 (Gu et al., 2008). The Xa10 gene was recentlycloned by map-based cloning and genetic transformation approaches(International Published Application No. WO 2012/033462). A functionaltarget sequence of AvrXa10, EBE_(AvrXa)10 was identified in the promoterof Xa10 (International Published Application No. WO 2012/033462). TheXa10 gene product, XA10, is functional in both monocots and dicots byinducing hypersensitive response (HR)-like cell death (unpublished).

The resistance specificity of TAL effector-dependent R gene to bacterialblight is determined by the R gene promoter rather than the R geneproducts (Gu et al., 2005). Meanwhile, the spectrum of TALeffector-dependent R genes to bacterial blight varies greatly, which isdepended on the availability of the avirulence TAL effectors in the X.oryzae pv. oryzae strains (Gu et al., 2004; Gu et al., 2008). Romer etal (2009) demonstrated that multiple functionally distinct DNA elementstargeted by separate TAL effectors retain their function and specificitywhen combined into one promoter. It is desired to generatebroad-spectrum resistance to bacterial blight in rice.

SUMMARY OF THE INVENTION

The present invention relates to synthetic promoter and a synthetic genewhich confers broad-spectrum disease resistance to Xanthomonands inplants. The present invention also relates to transgenic plantscontaining the synthetic gene and plants derived by crossing plants withsuch transgenic plants. More specifically, the synthetic promoter is asynthetic Xa10 promoter and the synthetic gene is a synthetic Xa10 genewhich contains the synthetic Xa10 promoter. The resistance is resistanceto bacterial blight and the plants are rice plants.

Thus, in a first aspect, the present invention provides a syntheticpromoter comprising a rice Xa10 promoter that has been modified tocontain multiple Effector Binding Elements (EBE) each of which binds adifferent transcription activator-like (TAL) effector. In oneembodiment, the synthetic promoter contains an EBE_(pthXo7) sequence. Inanother embodiment, the synthetic promoter contains an EBE_(pthXo1)sequence. In an additional embodiment, the synthetic promoter containsan EBE_(AvrXa10) sequence. In a further embodiment, the syntheticpromoter contains an EBE_(pthXo6) sequence. In another embodiment, thesynthetic promoter contains an EBE_(AvrXa27) sequence. In an additionalembodiment, the synthetic promoter contains all five of these EBEsequences. In one embodiment, the synthetic promoter is a synthetic minipromoter that contains one to five of the EBE sequences and the minimalportion of the rice Xa10 promoter to possess promoter activity. Inanother embodiment, the synthetic promoter is a synthetic full lengthXa10 that contains one to five of the EBE sequences. In one embodiment,the synthetic full length promoter contains the synthetic mini promoter.In a further embodiment, the synthetic promoter is any fragment of thesynthetic full length promoter that is larger than the synthetic minipromoter, that is contiguous to the 5′ end of the synthetic minipromoter, and that possesses promoter activity.

In one embodiment, the synthetic mini promoter comprises the sequenceset forth in SEQ ID NO:2. In another embodiment, the synthetic fulllength promoter comprises the sequence set forth in SEQ ID NO:10. In afurther embodiment, the synthetic promoter comprises nucleotides2208-2456 and any number of nucleotides 5′ to the synthetic minipromoter and contiguous to the synthetic mini promoter. In oneembodiment, the EBE_(pthXo7) sequence comprises the sequence set forthin SEQ ID NO:5. In another embodiment, the EBE_(pthXo1) sequencecomprises the sequence set forth in SEQ ID NO:6. In an additionalembodiment, the EBE_(AvrXa10) sequence comprises the sequence set forthin SEQ ID NO:7. In a further embodiment, the EBE_(pthXo6) sequencecomprises the sequence set forth in SEQ ID NO:8. In another embodiment,the EBE_(AvrXa27) sequence comprises the sequence set forth in SEQ IDNO:9.

In a second aspect, the present invention provides a synthetic Xa10 genewhich comprises a synthetic promoter of the present invention operablylinked to a rice Xa10 sequence. In one embodiment, the start codon ofthe rice Xa10 sequence is contiguous to the 3′ end of the syntheticpromoter. In another embodiment, the rice Xa10 sequence is the codingsequence of the rice Xa10 protein. In a further embodiment, the Xa10sequence is a genomic sequence encoding the rice Xa10 protein. Inanother embodiment, the rice Xa10 sequence is the coding sequence plusthe ′3 UTR containing the terminator. In a further embodiment, the riceXa10 terminator (nucleotides 382-759 of SEQ ID NO:15) can be replaced byother terminators well known to the skilled artisan, such as a NOSterminator, a 35S terminator and a Xa27 terminator. In one embodiment,the coding sequence comprises the coding sequence set forth in SEQ IDNO:13. In another embodiment, a genomic sequence encoding the rice Xa10protein comprises the sequence set forth in SEQ ID NO:15 which includesthe coding sequence (nucleotides 1-381) and the terminator (nucleotides382-759). In a further embodiment, a genomic sequence encoding the riceXa10 protein comprises the sequence set forth in SEQ ID NO:16 whichincludes the coding sequence (nucleotides 1-381), the terminator(nucleotides 382-759) and further 3′ UTR (nucleotides 760-1193). Inanother embodiment, a genomic sequence encoding the rice Xa10 proteincomprises the sequence set forth in SEQ ID NO:17 which includes thecoding sequence (nucleotides 1-381), the terminator (nucleotides382-759) and further 3′ UTR (nucleotides 760-2215). In anotherembodiment, the rice Xa10 sequence comprises the sequence set forth innucleotides 1-759 of SEQ ID NO:17 plus any number of nucleotides 3′ ofthe terminator in SEQ ID NO:17 that are contiguous to the terminator. Inone embodiment, the sequence of a synthetic Xa10 gene is set forth inSEQ ID NO:11. In another embodiment, the sequence of a synthetic Xa10gene is set forth in SEQ ID NO:12.

In a third aspect, the present invention provides a vector comprising asynthetic Xa10 gene described herein. The present invention alsoprovides a plant cell comprising the vector and a transgenic planthaving broad-spectrum resistance to bacterial blight comprising theplant cell. In one embodiment, the plant cell is a rice cell. In anotherembodiment, the transgenic plant is a transgenic rice plant.

In a fourth aspect, the present invention provides a method of making atransgenic plant having broad-spectrum resistance to bacterial blight.In accordance with the present invention, the method comprisestransfecting a synthetic Xa10 gene described herein or a vectorcomprising a synthetic Xa10 gene described herein into a plant cell orinto plant cells and producing a transgenic plant from the transfectedplant cell or transfected plant cells. In accordance with the presentinvention, the synthetic Xa10 gene is expressed in the transgenic plant.Transfecting the synthetic Xa10 gene or vector into a plant cell or intoplant cells is also sometimes referred to herein as transforming a plantcell or plant cells with the synthetic Xa10 gene or vector. In oneembodiment, the plant cell or cells is a rice cell or cells.

In a fifth aspect, the present invention provides a transgenic planthaving at least one copy of a synthetic Xa10 gene described hereinstably incorporated into its genome. In one embodiment, the transgenicplant contains two copies of the synthetic Xa10 gene. In anotherembodiment, the transgenic plant contains three copies of the syntheticXa10 gene. In an additional embodiment, the transgenic plant containsfour copies of the synthetic Xa10 gene. In a further embodiment, thetransgenic plant contains five copies of the synthetic Xa10 gene. Inanother embodiment, the transgenic plant contains six copies of thesynthetic Xa10 gene. In one embodiment, the plant is a rice plant. Inanother embodiment, the transgenic rice plant is referred to herein asthe L2 plant or L2 line. The present invention also provides for a plantthat contains a synthetic Xa10 gene described herein stably incorporatedinto its genome that is derived by crossing a transgenic plant describedherein or its progeny with a second plant and selecting progeny thatcontain the synthetic Xa10 gene. In accordance with the presentinvention, the transgenic plant or plants of the present invention canbe used in conventional breeding programs. In one embodiment, thetransgenic plant used in a breeding program is the transgenic rice plantreferred to herein as the L2 plant or L2 line or progeny thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the gene organization of Xa10^(E5). The nucleotidesequences of EBE_(pthXo7), EBE_(pthXo1), EBE_(AvrXa10), EBE_(pthXo6) andEBE_(AvrXa27) in the promoter of Xa10 gene are shown in bold and italicletters. The nucleotide sequences of the 5′ untranslated region ofXa10^(E5) are shown in lower letters. The restriction sites of BamHI,NruI and XbaI, and the size of DNA restriction fragments are indicated.Map was not drawn to scale. Xa10 ORF, open reading frame of the Xa10gene. The sequences shown in FIG. 1 are as follow: full length sequence:SEQ ID NO:1; Xa10^(E5) mini promoter (does not include Xa10 ORF): SEQ IDNO:2; Xa10 partial ORF shown: SEQ ID NO:3; Xa10 partial peptide shown:SEQ ID NO:4; EBE_(PthXo7): SEQ ID NO:5; EBE_(PthXo1): SEQ ID NO:6;EBE_(AvrXa10): SEQ ID NO:7; EBE_(PthXo6): SEQ ID NO:8; EBE_(AvrXa27):SEQ ID NO:9.

FIGS. 2A-2C show Southern blot analysis of transgenic plants of L2 line.FIG. 2A: Southern blot analysis of T₁ plants of L2 detected with Hptprobe. FIG. 2B: Southern blot analysis of T₂ plants of L2 detected withHpt probe. FIG. 2C: Southern blot analysis of T₂-36 and its T₃ progenydetected with Xa10 probe. About 2 μg of DNA samples were digested withrestriction enzymes BamHI and XbaI. The phenotypes of plants were shownbelow the images of Southern blot analyses. Arrows indicate the bandsthat co-segregated with the resistant phenotype. NB, Nipponbare.

FIG. 3 shows bacterial blight phenotype of L2 plants. Six-week-old T₃plants carrying homozygous Xa10^(E5), which was derived from T₂-36 plantof L2, were inoculated with X. oryzae pv. oryzae strain 1947 expressingTAL effectors AvrXa10, AvrXa27, pthXo1, pthX06 pthXo7 or empty vectorpHM1. Image was taken at 2 weeks after inoculation. Nipponbare (NB) wasused as the susceptible control.

FIGS. 4A and 4B show the induction of Xa10 in IRBB10A and L2 plants uponinoculation with X. oryzae pv. oryzae strains. FIG. 4A: Relativeexpression of Xa10 in IRBB10A and L2 plants. Xa10 transcripts weredetermined by qRT-PCR at 24 hpi. The expression of Xa10 in IRBB10Ainoculated 1947(pHM1avrXa10) was set as “1”. The rice ubiquitin gene 1(Ubi1) was used as an internal control. FIG. 4B: PCR products afteramplification with real-time RT-PCR. Samples in FIGS. 4A and 4B: 1,IRBB10A plants inoculated with water (mock inoculation); 2, IRBB10Aplants inoculated with 1947; 3, IRBB10A plants inoculated with1947(pHM1avrXa10); 4, L2 plants inoculated with water (mockinoculation); 5, L2 plants inoculated with 1947; 6, L2 plants inoculatedwith 1947(pHM1avrXa10); 7, L2 plants inoculated with 1947(pHM1avrXa27);8, L2 plants inoculated with 1947(pHM1pthXo1); 9, L2 plants inoculatedwith 1947(pHM1pthXo6); 10, L2 plants inoculated with 1947(pHM1pthXo7).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to synthetic promoter and a synthetic genewhich confers broad-spectrum disease resistance to Xanthomonands inplants. The present invention also relates to transgenic plantscontaining the synthetic gene and plants derived by crossing plants withsuch transgenic plants. More specifically, the synthetic promoter is asynthetic Xa10 promoter and the synthetic gene is a synthetic Xa10 genewhich contains the synthetic Xa10 promoter. The resistance is resistanceto bacterial blight and the plants are rice plants.

By “isolated” is meant a biological molecule free from at least some ofthe components with which it naturally occurs.

As used herein, “gene” refers to a nucleic acid sequence thatencompasses a 5′ promoter region associated with the expression of thegene product, any intron and exon regions and 3′ or 5′ untranslatedregions associated with the expression of the gene product.

As used herein, “genotype” refers to the genetic constitution of a cellor organism.

As used herein, “phenotype” refers to the detectable characteristics ofa cell or organism, which characteristics are the manifestation of geneexpression.

The terms “polynucleotide,” “nucleotide sequence,” and “nucleic acid”are used to refer to a polymer of nucleotides (A, C, T, U, G, etc. ornaturally occurring or artificial nucleotide analogues), e.g., DNA orRNA, or a representation thereof, e.g., a character string, etc.,depending on the relevant context. A given polynucleotide orcomplementary polynucleotide can be determined from any specifiednucleotide sequence.

A nucleic acid or polypeptide is “recombinant” when it is artificial orengineered, or derived from an artificial or engineered protein ornucleic acid. For example, a polynucleotide that is inserted into avector or any other heterologous location, e.g., in a genome of arecombinant organism, such that it is not associated with nucleotidesequences that normally flank the polynucleotide as it is found innature is a recombinant polynucleotide. A protein expressed in vitro orin vivo from a recombinant polynucleotide is an example of a recombinantpolypeptide. Likewise, a polynucleotide sequence that does not appear innature, for example a variant of a naturally occurring gene, isrecombinant.

The term “nucleic acid construct” or “polynucleotide construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or which has been modified tocontain segments of nucleic acids in a manner that would not otherwiseexist in nature. The term nucleic acid construct is synonymous with theterm or “expression cassette” when the nucleic acid construct containsthe control sequences required for expression of a sequence of thepresent invention. A “vector” is another type of nucleic acid construct.The vector may be an expression vector, a replication vector or atransformation vector.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolynucleotide of the present invention. Each control sequence may benative or foreign to the polynucleotide sequence. At a minimum, thecontrol sequences include a promoter and transcriptional stop signals.The control sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences to the nucleotide sequence.

The term “operably linked” is defined herein as a configuration in whicha control sequence is appropriately placed at a position relative to thenucleotide sequence of the nucleic acid construct such that the controlsequence directs the expression of a polynucleotide of the presentinvention.

In the present context, the term “expression” includes transcription ofthe polynucleotide. In the present context, the term “expression vector”covers a DNA molecule, linear or circular, that comprises apolynucleotide of the invention, and which is operably linked toadditional segments that provide for its transcription.

“Protein modifications” are provided by the present invention whichinclude one or more amino acid substitutions. Substitutional variantstypically contain the exchange of one amino acid for another at one ormore sites within the protein, and may be designed to modulate one ormore properties of the polypeptide, such as stability againstproteolytic cleavage, without the loss of other functions or properties.Amino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. Preferred substitutions areones which are conservative, that is, one amino acid is replaced withone of similar shape and charge. Conservative substitutions are wellknown to persons of ordinary skill in the art and typically include,though not exclusively, substitutions within the following groups:glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamicacid; asparagine, glutamine; serine, threonine; lysine, arginine; andtyrosine, phenylalanine.

Certain amino acids may be substituted for other amino acids in aprotein structure without appreciable loss of interactive bindingcapacity with structures such as, for example, antigen-binding regionsof antibodies or binding sites on substrate molecules or binding siteson proteins interacting with a polypeptide. Since it is the interactivecapacity and nature of a protein which defines that protein's biologicalfunctional activity, certain amino acid substitutions can be made in aprotein sequence, and its underlying DNA coding sequence, andnevertheless obtain a protein with like properties. In making suchchanges, the hydropathic index of amino acids may be considered. Theimportance of the hydrophobic amino acid index in conferring interactivebiological function on a protein is generally understood in the art.Alternatively, the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. The importance ofhydrophilicity in conferring interactive biological function of aprotein is generally understood in the art (See e.g. U.S. Pat. No.4,554,101). The use of the hydrophobic index or hydrophilicity indesigning polypeptides is further discussed in U.S. Pat. No. 5,691,198.

The term “plant” includes whole plants, shoot vegetativeorgans/structures (e.g. leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g. bracts, sepals, petals, stamens, carpels,anthers and ovules), seed (including embryo, endosperm, and seed coat)and fruit (the mature ovary), plant tissue (e.g. vascular tissue, groundtissue, and the like) and cells (e.g. guard cells, egg cells, trichomesand the like), and progeny of same. The class of plants that can be usedin the method of the invention is generally as broad as the class ofhigher and lower plants amenable to transformation techniques, includingangiosperms (monocotyledonous and dicotyledonous plants), gymnosperms,ferns, and multicellular algae. It includes plants of a variety ofploidy levels, including aneuploid, polyploid, diploid, haploid andhemizygous.

The term “heterologous” as used herein describes a relationship betweentwo or more elements which indicates that the elements are not normallyfound in proximity to one another in nature. Thus, for example, apolynucleotide sequence is “heterologous to” an organism or a secondpolynucleotide sequence if it originates from a foreign species, or, iffrom the same species, is modified from its original form. For example,a promoter operably linked to a heterologous coding sequence refers to acoding sequence from a species different from that from which thepromoter was derived, or, if from the same species, a coding sequencewhich is not naturally associated with the promoter (e.g. a geneticallyengineered coding sequence or an allele from a different ecotype orvariety). An example of a heterologous polypeptide is a polypeptideexpressed from a recombinant polynucleotide in a transgenic organism.Heterologous polynucleotides and polypeptides are forms of recombinantmolecules.

The term “transfecting” as used herein refers to the deliberateintroduction to a nucleic acid into a cell. Transfection includes anymethod known to the skilled artisan for introducing a nucleic acid intoa cell, including, but not limited to, Agrobacterium infection,ballistics, electroporation, microinjection and the like.

The term “broad-spectrum disease resistance” as used herein refers toresistance to a wide variety of strains of a causative agent. As anexample, a wide variety of strains may be strains of Xanthomonas oryzaepv. oryzae and the disease bacterial blight in rice.

TAL effectors from xanthomonads target host genes for promoting diseaseor triggering disease resistance. They bind to the effector bindingelements (EBEs) in the promoters of host genes and induce theirexpression. Xa10 is an R gene in rice that confer race-specificresistance to Xanthomonas oryzae pv. oryzae, the causal agent ofbacterial blight in rice. The Xa10 gene product, XA10, is functional inboth monocots and dicots by inducing hypersensitive response (HR)-likecell death.

In accordance with the present invention, novel resistance specificityand increased resistance spectrum of the Xa10 genes are accomplished bysynthesizing a modified Xa10 gene with an engineered promoter containing5 EBEs targeted by either virulent or avirulent TAL effectors (termedXa10^(E5) herein). The Xa10^(E5) gene was generated and used forproduction of transgenic rice. A stable transgenic rice line, L2, wasobtained. L2 had two copies of the Xa10^(E5) gene and conferred diseaseresistance to bacterial blight. The Xa10^(E5) gene in L2 plants wasspecifically induced by X. oryzae pv. oryzae strains expressing one ofthe corresponding TAL effectors and conferred broad-spectrum resistanceto 27 of the 28 field strains of X. oryzae pv. oryzae tested.

Thus, in a first aspect, the present invention provides a syntheticpromoter comprising a rice Xa10 promoter that has been modified tocontain multiple Effector Binding Elements (EBE) each of which binds adifferent transcription activator-like (TAL) effector. In oneembodiment, the synthetic promoter contains an EBE_(pthXo7) sequence. Inanother embodiment, the synthetic promoter contains an EBE_(pthXo1)sequence. In an additional embodiment, the synthetic promoter containsan EBE_(AvrXa10) sequence. In a further embodiment, the syntheticpromoter contains an EBE_(pthXo6) sequence. In another embodiment, thesynthetic promoter contains an EBE_(AvrXa27) sequence. In an additionalembodiment, the synthetic promoter contains all five of these EBEsequences. In one embodiment, the synthetic promoter is a synthetic minipromoter that contains one to five of the EBE sequences and the minimalportion of the rice Xa10 promoter to possess promoter activity. Inanother embodiment, the synthetic promoter is a synthetic full lengthXa10 that contains one to five of the EBE sequences. In one embodiment,the synthetic full length promoter contains the synthetic mini promoter.In a further embodiment, the synthetic promoter is any fragment of thesynthetic full length promoter that is larger than the synthetic minipromoter, that is contiguous to the 5′ end of the synthetic minipromoter, and that possesses promoter activity.

In one embodiment, the synthetic mini promoter comprises the sequenceset forth in SEQ ID NO:2. In another embodiment, the synthetic fulllength promoter comprises the sequence set forth in SEQ ID NO:10. In anadditional embodiment, the synthetic promoter comprises nucleotides2208-2456 of the sequence set forth in SEQ ID NO:10 and any number ofnucleotides that are contiguous and are contiguous to nucleotide 2208.In one embodiment, the synthetic promoter comprises nucleotides2200-2456 of the sequence set forth in SEQ ID NO:10. In anotherembodiment, the synthetic promoter comprises nucleotides 2100-2456 ofthe sequence set forth in SEQ ID NO:10. In an additional embodiment, thesynthetic promoter comprises nucleotides 1950-2456 of the sequence setforth in SEQ ID NO:10. In a further embodiment, the synthetic promotercomprises nucleotides 1500-2456 of the sequence set forth in SEQ IDNO:10. In another embodiment, the synthetic promoter comprisesnucleotides 1225-2456 of the sequence set forth in SEQ ID NO:10. In anadditional embodiment, the synthetic promoter comprises nucleotides1099-2456 of the sequence set forth in SEQ ID NO:10. In a furtherembodiment, the synthetic promoter comprises nucleotides 1438-2456 ofthe sequence set forth in SEQ ID NO:10. In another embodiment, thesynthetic promoter comprises nucleotides 987-2456 of the sequence setforth in SEQ ID NO:10. In an additional embodiment, the syntheticpromoter comprises nucleotides 542-2456 of the sequence set forth in SEQID NO:10. In a further embodiment, the synthetic promoter comprisesnucleotides 15-2456 of the sequence set forth in SEQ ID NO:10. Theseexamples of synthetic promoters are exemplary only and illustrate thatthe inventors contemplate any synthetic promoter comprising 250-2456contiguous nucleotides that must include nucleotides 2208-2246 of SEQ IDNO:10.

In one embodiment, the EBE_(pthXo7) sequence comprises the sequence setforth in SEQ ID NO:5. In another embodiment, the EBE_(pthXo1) sequencecomprises the sequence set forth in SEQ ID NO:6. In an additionalembodiment, the EBE_(AvrXa10) sequence comprises the sequence set forthin SEQ ID NO:7. In a further embodiment, the EBE_(pthXo6) sequencecomprises the sequence set forth in SEQ ID NO:8. In another embodiment,the EBE_(AvrXa27) sequence comprises the sequence set forth in SEQ IDNO:9.

In a second aspect, the present invention provides a synthetic Xa10 genewhich comprises a synthetic promoter of the present invention operablylinked to a rice Xa10 sequence. In one embodiment, the start codon ofthe rice Xa10 sequence is contiguous to the 3′ end of the syntheticpromoter. In another embodiment, the rice Xa10 sequence is the codingsequence of the rice Xa10 protein. In a further embodiment, the Xa10sequence is a genomic sequence encoding the rice Xa10 protein. Inanother embodiment, the rice Xa10 sequence is the coding sequence plusthe ′3 UTR containing the terminator. In a further embodiment, the riceXa10 terminator (nucleotides 382-759 of SEQ ID NO:15) can be replaced byother terminators well known to the skilled artisan, such as a NOSterminator, a 35S terminator and a Xa27 terminator. In one embodiment,the coding sequence comprises the coding sequence set forth in SEQ IDNO:13. In another embodiment, a genomic sequence encoding the rice Xa10protein comprises the sequence set forth in SEQ ID NO:15 which includesthe coding sequence (nucleotides 1-381) and the terminator (nucleotides382-759). In a further embodiment, a genomic sequence encoding the riceXa10 protein comprises the sequence set forth in SEQ ID NO:16 whichincludes the coding sequence (nucleotides 1-381), the terminator(nucleotides 382-759) and further 3′ UTR (nucleotides 760-1193). Inanother embodiment, a genomic sequence encoding the rice Xa10 proteincomprises the sequence set forth in SEQ ID NO:17 which includes thecoding sequence (nucleotides 1-381), the terminator (nucleotides382-759) and further 3′ UTR (nucleotides 760-2215). In anotherembodiment, the rice Xa10 sequence comprises the sequence set forth innucleotides 1-759 of SEQ ID NO:17 plus any number of nucleotides 3′ ofthe terminator in SEQ ID NO:17 that are contiguous to the terminator. Inone embodiment, the sequence of a synthetic Xa10 gene is set forth inSEQ ID NO:11. In another embodiment, the sequence of a synthetic Xa10gene is set forth in SEQ ID NO:12.

In one embodiment, the rice Xa10 sequence comprises the sequence setforth in nucleotides 1-381 of SEQ ID NO:17 plus any number ofnucleotides 3′ of the stop codon in SEQ ID NO:17 that are contiguous tothe stop codon. In one embodiment, the Xa10 sequence comprisesnucleotides 1-420 of the sequence set forth in SEQ ID NO:17. In anotherembodiment, the Xa10 sequence comprises nucleotides 1-625 of thesequence set forth in SEQ ID NO:17. In an additional embodiment, theXa10 sequence comprises nucleotides 1-852 of the sequence set forth inSEQ ID NO:17. In a further embodiment, the Xa10 sequence comprisesnucleotides 1-1178 of the sequence set forth in SEQ ID NO:17. In anotherembodiment, the Xa10 sequence comprises nucleotides 1-1643 of thesequence set forth in SEQ ID NO:17. In and additional embodiment, theXa10 sequence comprises nucleotides 1-2011 of the sequence set forth inSEQ ID NO:17. In a further embodiment, the Xa10 sequence comprisesnucleotides 1-2211 of the sequence set forth in SEQ ID NO:17. Theseexamples of Xa10 sequences are exemplary only and illustrate that theinventors contemplate any Xa10 sequence comprising 382-2215 contiguousnucleotides of SEQ ID NO:17 that must include nucleotides 1-381 of SEQID NO:17.

In one embodiment, the rice Xa10 sequence comprises the sequence setforth in nucleotides 1-759 of SEQ ID NO:17 which includes the codingsequence (nucleotides 1-381) and the terminator (nucleotides 382-759)plus any number of nucleotides 3′ of the terminator in SEQ ID NO:17 thatare contiguous to the terminator. In one embodiment, the Xa10 sequencecomprises nucleotides 1-820 of the sequence set forth in SEQ ID NO:17.In another embodiment, the Xa10 sequence comprises nucleotides 1-1095 ofthe sequence set forth in SEQ ID NO:17. In an additional embodiment, theXa10 sequence comprises nucleotides 1-1587 of the sequence set forth inSEQ ID NO:17. In a further embodiment, the Xa10 sequence comprisesnucleotides 1-2050 of the sequence set forth in SEQ ID NO:17. Theseexamples of Xa10 sequences are exemplary only and illustrate that theinventors contemplate any Xa10 sequence comprising the Xa10 codingsequence and terminator which comprises 760-2215 contiguous nucleotidesof SEQ ID NO:17 that must include nucleotides 1-759 of SEQ ID NO:17.

In a third aspect, the present invention provides a vector as describedherein comprising a synthetic Xa10 gene described herein. Such vectorsare well known to the skilled artisan or described further below. Thepresent invention also provides a plant cell comprising the vector and atransgenic plant having broad-spectrum resistance to bacterial blightcomprising the plant cell. In one embodiment, the plant cell is a ricecell. In another embodiment, the transgenic plant is a transgenic riceplant.

In a fourth aspect, the present invention provides a method of making atransgenic plant having broad-spectrum resistance to bacterial blight.In accordance with the present invention, the method comprisestransfecting a synthetic Xa10 gene described herein or a vectorcomprising a synthetic Xa10 gene described herein into a plant cell orinto plant cells and producing a transgenic plant from the transfectedplant cell or transfected plant cells. In accordance with the presentinvention, the synthetic Xa10 gene is expressed in the transgenic plant.Transfecting the synthetic Xa10 gene or vector into a plant cell or intoplant cells is also sometimes referred to herein as transforming a plantcell or plant cells with the synthetic Xa10 gene or vector. In oneembodiment, the plant cell or cells is a rice cell or cells. Suchmethods are well known to the skilled artisan or described furtherbelow.

In a fifth aspect, the present invention provides a transgenic planthaving at least one copy of a synthetic Xa10 gene described hereinstably incorporated into its genome. In one embodiment, the transgenicplant contains two copies of the synthetic Xa10 gene. In anotherembodiment, the transgenic plant contains three copies of the syntheticXa10 gene. In an additional embodiment, the transgenic plant containsfour copies of the synthetic Xa10 gene. In a further embodiment, thetransgenic plant contains five copies of the synthetic Xa10 gene. Inanother embodiment, the transgenic plant contains six copies of thesynthetic Xa10 gene. In one embodiment, the plant is a rice plant. Inanother embodiment, the transgenic rice plant is referred to herein asthe L2 plant or L2 line. The present invention also provides for a plantthat contains a synthetic Xa10 gene described herein stably incorporatedinto its genome that is derived by crossing a transgenic plant describedherein or its progeny with a second plant and selecting progeny thatcontain the synthetic Xa10 gene. In accordance with the presentinvention, the transgenic plant or plants of the present invention canbe used in conventional breeding programs. In one embodiment, thetransgenic plant used in a breeding program is the transgenic rice plantreferred to herein as the L2 plant or L2 line or progeny thereof.

Generally, the vector or expression cassette may additionally comprise aselectable marker gene for the selection of transformed cells.Selectable marker genes are utilized for the selection of transformedcells or tissues. Usually, the plant selectable marker gene will encodeantibiotic resistance, with suitable genes including at least one set ofgenes coding for resistance to the antibiotic spectinomycin, thestreptomycin phosphotransferase (spt) gene coding for streptomycinresistance, the neomycin phosphotransferase (nptII) gene encodingkanamycin or geneticin resistance, the hygromycin phosphotransferase(hpt or aphiv) gene encoding resistance to hygromycin, acetolactatesynthase (als) genes. Alternatively, the plant selectable marker genewill encode herbicide resistance such as resistance to thesulfonylurea-type herbicides, glufosinate, glyphosate, ammonium,bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D),including genes coding for resistance to herbicides which act to inhibitthe action of glutamine synthase such as phosphinothricin or basta(e.g., the bar gene). See generally, International Publication Nos. WO02/36782 and WO 2008/094127, U.S. Pat. No. 7,205,453 and U.S. PatentApplication Publication Nos. 2006/0218670, 2006/0248616, 2007/0143880and 2009/0100536, and the references cited therein. See also, Jeffersonet al. (1991); De Wet et al. (1987); Goff et al. (1990); Kain et al.(1995) and Chiu et al. (1996). This list of selectable marker genes isnot meant to be limiting. Any selectable marker gene can be used. Theselectable marker gene is also under control of a promoter operable inthe plant species to be transformed. Such promoters include thosedescribed in International Publication No. WO 2008/094127, U.S. PatentApplication Publication No. 2012/0245339, and the references citedtherein. See also, U.S. Patent Application Publication Nos. 2008/0313773and 2010/0199371 for an exemplification of additional markers that canbe used in accordance with the present invention.

Alternatively, the vector or expression cassette may additionallycomprise a Cre-lox recombination marker free system, such as describedherein. Such a system is useful for producing selection marker freetransgenic plants.

In preparing the vector or expression cassette, the various DNAfragments may be manipulated, so as to provide for the DNA sequences inthe proper orientation and, as appropriate, in the proper reading frame.Toward this end, adapters or linkers may be employed to join the DNAfragments or other manipulations may be involved to provide forconvenient restriction sites, removal of superfluous DNA, removal ofrestriction sites, or the like. For this purpose, in vitro mutagenesis,primer repair, restriction, annealing, resubstitutions, e.g. transitionsand transversions may be involved.

Once a nucleic acid, such as a synthetic Xa10 gene described herein, hasbeen cloned into a vector or an expression vector, it may be introducedinto a plant cell using conventional transformation procedures. The term“plant cell” is intended to encompass any cell derived from a plantincluding undifferentiated tissues such as callus and suspensioncultures, as well as plant seeds, pollen or plant embryos. Plant tissuessuitable for transformation include leaf tissues, root tissues,meristems, protoplasts, hypocotyls, cotyledons, scutellum, shoot apex,root, immature embryo, pollen, and anther. “Transformation” means thedirected modification of the genome of a cell by the externalapplication of recombinant DNA from another cell of different genotype,leading to its uptake and integration into the subject cell's genome. Inthis manner, genetically modified plants, plant cells, plant tissue,seed, and the like can be obtained.

The nucleic acids, vectors or constructs may be introduced into thegenome of the desired plant host by a variety of conventionaltechniques. Techniques for transforming a wide variety of higher plantspecies are well known and described in the technical and scientificliterature. Transformation protocols may vary depending on the type ofplant or plant cell, i.e., monocot or dicot, targeted fortransformation, as is well known to the skilled artisan. For example,the DNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as electroporation and microinjectionof plant cell protoplasts, or the DNA constructs can be introduceddirectly to plant tissue using ballistic methods, such as DNA particlebombardment. Alternatively, the DNA constructs may be combined withsuitable T-DNA flanking regions and introduced into a conventionalAgrobacterium tumefaciens host vector. The virulence functions of theAgrobacterium tumefaciens host will direct the insertion of theconstruct and adjacent marker into the plant cell DNA when the cell isinfected by the bacteria. Thus, any method, which provides for effectivetransformation/transfection may be employed. See, for example, U.S. Pat.Nos. 7,241,937, 7,273,966 and 7,291,765 and U.S. Patent ApplicationPublication Nos. 2007/0231905 and 2008/0010704 and references citedtherein. See also, International Published Application Nos. WO2005/103271, WO 2005/017158, WO 2008/094127, WO 2012/033462 andreferences cited therein.

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotype,e.g., a transgenic plant. A “transgenic plant” is a plant into whichforeign DNA has been introduced. A “transgenic plant” encompasses alldescendants, hybrids, and crosses thereof, whether reproduced sexuallyor asexually, and which continue to harbor the foreign DNA. Regenerationtechniques rely on manipulation of certain phytohormones in a tissueculture growth medium, typically relying on a biocide and/or herbicidemarker which has been introduced together with the desired nucleotidesequences. See for example, International Published Application No. WO2008/094127 and references cited therein.

The foregoing methods for transformation are typically used forproducing a transgenic variety in which the expression cassette isstably incorporated. After the expression cassette is stablyincorporated in transgenic plants, it can be transferred to other plantsby sexual crossing. In one embodiment, the transgenic variety could thenbe crossed, with another (non-transformed or transformed) variety, inorder to produce a new transgenic variety. Alternatively, a genetictrait which has been engineered into a particular cotton line using theforegoing transformation techniques could be moved into another lineusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite variety into anelite variety, or from a variety containing a foreign gene in its genomeinto a variety or varieties which do not contain that gene. As usedherein, “crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context. Any of a number of standardbreeding techniques can be used, depending upon the species to becrossed.

Once transgenic plants of this type are produced, the plants themselvescan be cultivated in accordance with conventional procedures. Transgenicseeds can, of course, be recovered from the transgenic plants. Theseseeds can then be planted in the soil and cultivated using conventionalprocedures to produce transgenic plants. The cultivated transgenicplants will express the synthetic Xa10 gene to provide broad-spectrumresistance to bacterial blight. The cultivated transgenic plants canalso be used in conventional breeding programs to derive additionalplants that will express the synthetic Xa10 gene to providebroad-spectrum resistance to bacterial blight.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989,Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York);Green and Sambrook, 2012, Molecular Cloning, 4th Ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); Ausubel et al., 1992,Current Protocols in Molecular Biology (John Wiley & Sons, includingperiodic updates); Glover, 1985, DNA Cloning (IRL Press, Oxford);Russell, 1984, Molecular biology of plants: a laboratory course manual(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Anand,Techniques for the Analysis of Complex Genomes, (Academic Press, NewYork, 1992); Guthrie and Fink, Guide to Yeast Genetics and MolecularBiology (Academic Press, New York, 1991); Harlow and Lane, 1988,Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds.1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc.,1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, APractical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology (Academic Press, Inc., N.Y.); Methods In Enzymology, Vols.154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker, eds., Academic Press, London,1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir andC. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition,Blackwell Scientific Publications, Oxford, 1988; Fire et al., RNAInterference Technology: From Basic Science to Drug Development,Cambridge University Press, Cambridge, 2005; Schepers, RNA Interferencein Practice, Wiley-VCH, 2005; Engelke, RNA Interference (RNAi): The Nuts& Bolts of siRNA Technology, DNA Press, 2003; Gott, RNA Interference,Editing, and Modification: Methods and Protocols (Methods in MolecularBiology), Human Press, Totowa, N.J., 2004; Sohail, Gene Silencing by RNAInterference: Technology and Application, CRC, 2004.

EXAMPLES

The present invention is described by reference to the followingExamples, which is offered by way of illustration and is not intended tolimit the invention in any manner. Standard techniques well known in theart or the techniques specifically described below were utilized.

Example 1 Materials and Methods

TAL Effectors and Xanthomonas oryzae pv. Oryzae (Xoo) Strains:

TAL effector genes, avrXa10, avrXa27, pthXo1, pthXo6 and pthXo7, havebeen reported previously (Hopkins et al., 1992; Gu et al., 2005; Yang etal., 2006; Sugio et al., 2007). TAL effector genes were cloned intovector pBluescript and placed under lacZ promoter. The intermediateconstruct were then fused with cosmid vector pHM1 (GenBank Accession No.EF059993) at the Hind III site. Cosmid constructs carrying the TALeffectors were introduced into X. oryzae pv. oryzae strain 1947 byelectroporation. Twenty-eight field strains of X. oryzae pv. oryzae,which were collected from 11 countries were used in this study.

Plants and Growth Conditions:

IRBB10A is an improved near-isogenic line (NIL) of Xa10 in the IR24genetic background (Gu et al., 2008). Nipponbare is a japonica ricecultivar. Rice plants, including transgenic plants, were grown in agreenhouse at a temperature of 32° C. and 25° C. for 12.5 h (light) and11.5 h (dark), respectively.

Binary Construct:

Binary construct pCXA10E5 carrying Xa10^(E5) (FIG. 1) was made based onpCAS4671, which harbors 4671-bp AvrII-SacI genomic clone of Xa10 inbinary vector pC1300. A 661-bp synthetic BamHI-NruI DNA fragmentcontaining EBE_(pthXo7), EBE_(pthXo1), EBE_(AvrXa10), EBE_(pthXo6) andEBE_(AvrXa27) were used to replace the 657-bp BamHI-NruI fragment in thenative promoter of Xa10 gene. The pCXA10E5 was introduced intoAgrobacterium tumefaciens strain AGL1 by electroporation.

Rice Transformation:

Agrobacterium-mediated transformation of Nipponbare was carried outusing the method described previously (Hiei et al., 1994) with slightmodification. Briefly, vigorously growing embryogenic calli derived fromthe scutellum of mature embryos was co-cultured with A. tumefaciensstrains harbouring binary plasmids for 2 days. After co-culture, therice tissues were washed and then cultured on selection mediumcomprising NB basal medium (NB₀ medium; contains the macronutrientsdescribed by Chu (1975) and the micronutrients described by Gamborg etal. (1968)) containing 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D),250 mg/L cefotaxime and 50 mg/L hygromycin at 26° C. in the dark. After4 weeks selection, the hygromycin-resistant calli were excised fromco-cultivated callus and transferred to regeneration medium comprisingNB₀ medium containing 2.0 mg/L kinetin (KT), 0.02 mg/Lα-naphthaleneacetic acid (NAA) and 50 mg/L hygromycin. Three weeks laterthe regenerated hygromycin-resistant plantlets were transferred toplantlet medium comprising ½ strength MS basal medium (½ MS₀ medium; MSbasal medium defined by Murashige and Skoog (1962)) containing 50 mg/Lhygromycin for shoot and root elongation. Regenerated plantlets weresubsequently transplanted to the soil in pots and grown in a greenhouse.

Bacterial Blight Inoculation and Disease Scoring:

Bacterial inoculation was carried out using the leaf-clipping method(Kauffman et al., 1973). Briefly, X. oryzae pv. oryzae strains weregrown on PSA medium (10 g/l peptone, 10 g/l sucrose, 1 g/l glutamicacid, 16 g/l bacto-agar, and pH 7.0) for 2 days at 28° C. The bacterialcells were collected and re-suspended in sterile water at an opticaldensity of 0.5 at OD600. The bacterial cell suspension was applied tothe two youngest, but fully expanded, leaves of each tiller by clipping5-6 cm, from the tip of the leaf using a pair of scissors dipped in theinoculum. Lesion length (LL) was measured 2 weeks after inoculation. Thedisease symptom was scored as resistant (R, LL≦3.0 cm), moderatelyresistant (MR, 3.0 cm<LL≦6.0 cm), moderately susceptible (MS, 6.0cm<LL≦9.0 cm) and susceptible (S, LL>9.0 cm) (Gu et al., 2004).

Southern Blot Analysis:

Rice genomic DNA was isolated from leaf tissues according to theprocedures described previously (Dellaporta et al., 1983). About 2 μg ofDNA was digested with the appropriate restriction enzymes, separated on0.8% agarose gel and blotted to Hybond™−N+nylon membrane (AmershamBiosciences, GE healthcare, USA). DNA hybridization was conductedaccording to standard procedures (Sambrook et al., 1989). DNA probeswere labeled with DIG system (Roche Applied Science, USA). The DNA probefor the Hpt gene was amplified by PCR using DNA primers Hpt-F(5′AAAAAGCCTGAACTCACC GCG3′; SEQ ID NO:18) and Hpt-R(5′TACTTCTACACAGCCATCGGT3′; SEQ ID NO:19). The DNA probe for the Xa10gene was amplified by PCR using DNA primers Xa10-F(5′CACGGGCCCCCTCCTGTTTGC3′; SEQ ID NO:20) and Xa10-R (5′CCTCGTCGTCTTCACCAATGCAG3′; SEQ ID NO:21). The primers used for recovering theXa10^(E5) gene from the L2 line were Seq F5(5′CTAGGTTTATTGGCTGAGCAATG3′; SEQ ID NO:22); Seq R5(5′TCTAACTGTCGCCGATCTGCTG3′; SEQ ID NO:23), Seq F4 (5′TTCTTCTTCCTTCCTCTCTCTAC3′; SEQ ID NO:24), Seq R4 (5′TCACTCGCCATGTCTCTACTTC3′; SEQ IDNO:25), Seq F3 (5′AGGAAAGGTGAGAAGGGAATTG3′; SEQ ID NO:26), Seq R3(5′GTTT AGCTCGACTTTCGACCCAG3′; SEQ ID NO:27), 4686F(5′CTGGGTCGAAAGTCGAGCT AAAC3′; SEQ ID NO:28), Xa10H5R(5′CCATCGTGATATATATGGGCCTCAGACG3′; SEQ ID NO:29), Seq F1(5′GCCATACTCTTCTACCTCTCTG3′; SEQ ID NO:30), Seq R1(5′TTATTCTTTTCATATCATTTGATTC3′; SEQ ID NO:31), Seq F2 (5′GTAAAACTATATGTGTACATGG3′; SEQ ID NO:32) and Seq R2 (5′TCCTCGCTAAATTAAAGTGATG3′; SEQID NO:33).

Quantitative reverse-transcription PCR (qRT-PCR): qRT-PCR was carriedout in accordance with the procedures described previously (Gu et al.,2011) using a CFX96 real-time PCR system (Bio-Rad). Total RNA wasextracted from rice leaves at 24 hours post inoculation (hpi) with X.oryzae pv. oryzae strains. The total RNA samples were first treated withDNase I, and then reversely transcribed to first-strand cDNAs usingiScript cDNA synthesis kit (Bio-Rad) according to the manufacturer'sinstructions. To ensure maximum specificity and efficiency duringquantitative PCR, oligo primers were further tested for linearity byconstructing standard curves on five or six serial 10-fold dilutions. Astandard reaction mixture (15 μl) contained 2 μl cDNA template, 1×SsoFast EvaGreen supermix (Bio-Rad) and 500 nM forward and reverseprimers. The PCR reaction was conducted at an initial denaturing step of98° C. for 3 min, followed by 40 cycles of 98° C. for 5 s, 55° C. for 5s. A melting-curve reaction was subsequently performed for 5 s, startingat 75° C. with 0.2° C. increments. PCR product specificity was confirmedby melting-curve analysis and separation on a 2% agarose gel to ensurethat the PCR reactions were free of primer dimers. The rice ubiquitingene 1 (Ubi1) was used as an internal reference to normalize therelative amount of total RNA for all samples. The qRT-PCR experimentswere repeated for three times. Rice Ubi gene was used as an internalcontrol. The specific primers for Xa10 were 10RTF2(5′GGCATCATCTTCTCCGGCG3′; SEQ ID NO:34) and 10RTR2(5′GCAGCTATACGGGCATAAG3′; SEQ ID NO:35). The specific primers for Ubi1were RBQ3 (5′CCAGTAAGTCCTCAGCCATG3′; SEQ ID NO:36) and RBQ4 (5′TTTCAGACACCATCAAACCAG3′; SEQ ID NO:37).

Example 2 Production of Transgenic Lines Carrying Xa10^(E5)

Transgenic rice plants were produced by Agrobacterium-mediatedtransformation of Nipponbare (Hiei et al., 1994). After one monthsubculture of co-cultivated calli on selective medium, 1,896hyromycin-resistant calli were obtained. However, most of thehyromycin-resistant calli turned brown and eventually died after theywere transferred onto regeneration medium and cultured under light. Onlya few of the hyromycin-resistant calli remained healthy and eventuallyregenerated transgenic plantlets. Finally, 8 independent transgeniclines were obtained. Some lines might have more than one T₀ plantsderived from a single transformed cell. T₀ plants were transplanted tosoil and grown in greenhouse.

Five-week-old T₀ plants were inoculated with X. oryzae pv. oryzae strain1947 expressing AvrXa10. Disease evaluation at two weeks after bacterialblight inoculation indicated that only T₀ plants from two lines, L2 andL5, conferred complete resistance to 1947(pHM1AvrXa10). Other T₀ plantswere susceptible to the Xa10-incompatible strain. Eight T₀ plants of L2were obtained and they all displayed normal morphological phenotype andgrowth duration compared to that of wild-type plants. One T₀ plant of L5was obtained and the plant showed slightly stress-related phenotypes,such as stiff leaves and longer growth duration, compared to that ofwild-type. The stress-related phenotypes were more severe in homozygotein the T₂ or T₃ generations. L5 was abandoned for further study.

There were at least 6 copies of T-DNA in the T₀ plants of L2 detected bySouthern blot analysis using Hpt probe. They could be separated in theT₁ generation (FIG. 2A). However, only one copy of T-DNA, which showedthe hybridized band at about 5.0 kb detected by Hpt probe, carriedfunctional Xa10^(E5) gene and co-segregated with the resistantphenotype. T₂ plants that only carried the functional Xa10^(E5) genewere obtained from T₂ progeny of T₁-80 (FIG. 2B). One of the T₂ plants,T₂-36 carried homozygous Xa10^(E5) gene. T₂-36 had two copies ofXa10^(E5) gene with hybridized bands at about 5.2 kb and 7.4 kb,respectively, detected by Xa10 probe (FIG. 2C). The two hybridized bandswere greater than the expected 2866-bp band of the BamHI-XbaI fragmentfrom the Xa10^(E) gene (FIG. 1). In addition, the two copies ofXa10^(E5) gene co-segregated in the subsequent generation (FIG. 2C). Theresults indicated that the two copies of Xa10^(E5) gene were parts ofthe two truncated T-DNAs which were integrated into Nipponbare genome inthe same or closely-related location after illegitimate T-DNAintegration. The intact Xa10^(E5) gene, consisting of the Xa10^(E5)promoter (2456 bp), the Xa10 coding region (381-bp), the Xa10 terminator(378 bp) and a 434-bp 3′ region flanking the Xa10 terminator, wererecovered from the T₂-36 plant and its progeny by PCR amplification andDNA sequencing. The genomic sequence of Xa10^(E5) recovered from L2 isset forth in SEQ ID NO:12. The regions of the genomic sequence ofXa10^(E5) are as follows: nucleotides 1-2456 is a Xa10^(E5) promoter;nucleotides 2457-2837 is the Xa10 ORF; nucleotides 2838-3215 is a Xa10terminator; and nucleotides 3216-3649 is a 3′ region flanking the Xa10terminator. T₂-36 progeny were still designated as L2 that carriesfunctional Xa10^(E5) genes for the further studies.

Example 3 Xa10^(E5) was Induced by X. oryzae pv. Oryzae StrainsExpressing the Corresponding TAL Effectors

L2 plants conferred resistance to X. oryzae pv. oryzae strains1947(pHM1avrXa10), 1947(pHM1avrXa27), 1947(pHM1pthXo1), 1947(pHM1pthXo6) and 1947(pHM1pthXo7), whereas Nipponbare plants weresusceptible to all these strains (FIG. 3). In control experiments, bothL2 and Nipponbare were susceptible to X. oryzae pv. oryzae strain 1947(FIG. 3).

qRT-PCR analysis indicated that the Xa10^(E5) gene in L2 plants wasactivated after inoculation with incompatible X. oryzae pv. oryzaestrains (FIG. 4). The expression levels of Xa10 transcripts in L2 plantsinoculated with 1947(pHM1avrXa27), 1947(pHM1pthXo1), 1947(pHM1pthXo6) or1947(pHM1pthXo7) were comparable to that of Xa10 transcripts in IRBB10Aplants inoculated with 1947(pHM1avrXa10) (FIG. 4). The expression levelof Xa10 transcripts in L2 plants inoculated with 1947(pHM1avrXa10) wasonly 13% to that of Xa10 transcripts in IRBB10A plants inoculated with1947(pHM1avrXa10) (FIG. 4), which was still sufficient to providecomplete resistance to the incompatible strain (FIG. 3). Likewise inIRBB10A plants, the Xa10 transcript was not detected in the L2 plantsafter mock inoculation or inoculation with 1947(pHM1) (FIG. 4).

Example 4 Xa10^(E5) Conferred Broad Spectrum Resistance to MultipleField Strains Collected from Different Countries

Twenty-eight field strains of X. oryzae pv. oryzae (Xoo) collected fromdifferent rice growing countries around the world (Table 1) were used totest the resistant spectrum of Xa10^(E5) in L2 plants to bacterialblight disease. Non-transgenic Nipponbare plants were susceptible ormoderate susceptible to most of the X. oryzae pv. oryzae strains testedand only showed moderate resistance to GD1358 from China and CIAT1185from Columbia (Table 1). IRBB10A is an improved near-isogenic line ofXa10 in IR24 genetic background (Gu et al., 2008). IRBB10A only showedcomplete resistance to PXO86(R2) and PXO112(R5), and moderate resistanceto Aust-2031 and Aust-R3 (Table 1 and (Gu et al., 2008)). The L2 plantsconferred high and broad-spectrum resistance to 27 Xoo strains testedexcept for 1947, an X. oryzae pv. oryzae strain from Africa (Table 1).

TABLE 1 Resistant Spectrum of Xa10^(E5) L2 to Xoo Strains Collected fromDifferent Countries Lesion length and resistance score^(a) Xoo StrainOrigin IR24 IRBB10A NB L2 1947 Africa 27.0 ± 6.3 (S) 28.6 ± 4.8 (S) 15.8± 3.8 (S) 10.1 ± 0.9 (S)  Aust-2031 Australia  5.5 ± 1.4 (MR)  4.4 ± 1.6(MR)  9.4 ± 1.8 (S) 0.2 ± 0.1 (R) Aust-R3 Australia  5.7 ± 1.5 (MR)  6.8± 2.0 (MR) 10.9 ± 4.2 (S) 0.1 ± 0.0 (R) GD1358 China 19.0 ± 7.3 (S) 21.1± 3.6 (S)  4.0 ± 1.6 (MR) 0.1 ± 0.0 (R) HB17 China 25.3 ± 3.8 (S) 24.7 ±4.0 (S) 21.2 ± 2.0 (S) 0.1 ± 0.0 (R) HB21 China 19.6 ± 2.6 (S) 15.2 ±3.5 (S) 20.8 ± 1.7 (S) 0.1 ± 0.0 (R) HLJ72 China 10.0 ± 2.5 (S)  9.8 ±3.0 (S) 12.1 ± 1.3 (S) 0.1 ± 0.0 (R) JS49-6 China 24.6 ± 3.6 (S) 21.6 ±4.0 (S) 18.3 ± 2.6 (S) 0.1 ± 0.0 (R) LN57 China 22.1 ± 2.8 (S) 25.4 ±3.5 (S) 20.1 ± 4.8 (S) 0.1 ± 0.0 (R) NX42 China 23.3 ± 4.7 (S) 23.2 ±4.8 (S) 20.6 ± 2.2 (S) 0.1 ± 0.0 (R) ZHE173 China 21.8 ± 3.9 (S) 23.0 ±4.5 (S) 14.5 ± 2.8 (S) 0.1 ± 0.0 (R) CIAT1185 Columbia 18.1 ± 4.8 (S)13.6 ± 3.5 (S)  4.6 ± 2.1 (MR) 0.1 ± 0.0 (R) A3842 India 22.4 ± 3.8 (S)21.5 ± 3.9 (S) 16.6 ± 3.4 (S) 0.1 ± 0.0 (R) A3857 India 19.1 ± 2.7 (S)18.3 ± 4.6 (S) 19.0 ± 1.9 (S) 0.1 ± 0.0 (R) IXO56 Indonesia 24.1 ± 5.2(S) 23.9 ± 3.6 (S) 13.3 ± 2.5 (S) 0.1 ± 0.0 (R) H75373 Japan 25.7 ± 5.7(S) 24.4 ± 4.3 (S) 17.9 ± 2.0 (S) 0.1 ± 0.0 (R) T7174 Japan 23.3 ± 3.6(S) 19.1 ± 3.0 (S) 19.7 ± 2.1 (S) 0.2 ± 0.1 (R) JW89011 Korea 23.8 ± 5.1(S) 26.6 ± 5.1 (S) 16.7 ± 2.3 (S) 0.1 ± 0.0 (R) K202 Korea 25.6 ± 3.9(S) 26.3 ± 4.3 (S) 12.3 ± 2.3 (S) 0.1 ± 0.0 (R) NXO260 Nepal 22.4 ± 3.4(S) 22.4 ± 4.5 (S) 16.6 ± 2.0 (S) 0.1 ± 0.0 (R) PXO86(R2) Philippines21.9 ± 3.1 (S)  0.2 ± 0.1 (R) 12.7 ± 2.1 (S) 0.1 ± 0.0 (R) PXO79(R3)Philippines 23.1 ± 4.3 (S) 20.2 ± 4.1 (S)  6.7 ± 2.3 (MS) 0.1 ± 0.0 (R)PXO71(R4) Philippines 23.1 ± 3.3 (S) 23.2 ± 4.7 (S) 15.9 ± 1.8 (S) 0.1 ±0.0 (R) PXO112(R5) Philippines 13.4 ± 2.5 (S)  0.1 ± 0.0 (R) 11.8 ± 3.1(S) 0.1 ± 0.0 (R) PXO113(R4) Philippines 18.5 ± 2.1 (S) 18.6 ± 3.7 (S)11.0 ± 3.6 (S) 0.1 ± 0.0 (R) PXO99(R6) Philippines 23.4 ± 3.3 (S) 23.2 ±2.9 (S) 14.2 ± 3.1 (S) 0.1 ± 0.0 (R) 2 Thailand 25.4 ± 3.6 (S) 26.4 ±3.9 (S) 18.6 ± 4.4 (S) 0.1 ± 0.0 (R) R7 Thailand 10.0 ± 6.5 (S)  6.9 ±2.7 (MS)  9.9 ± 2.3 (S) 0.1 ± 0.0 (R) ^(a)Six-Week-old rice plants wereinoculated with X. oryzae pv. oryzae strains. Lesion length and diseasephenotype of the inoculated plants were scored at two weeks afterinoculation. For disease scoring: R, resistant, lesion length ≦3.0 cm;MR, moderately resistant, lesion length >3.0 cm and ≦6.0 cm; MS,moderately susceptible, lesion length >6.0 cm and ≦9.0 cm; S,susceptible, lesion length >9.0 cm.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the invention and does not pose a limitation on the scope ofthe invention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. Embodiments of this inventionare described herein, including the best mode known to the inventors forcarrying out the invention. Variations of those embodiments may becomeapparent to those of ordinary skill in the art upon reading theforegoing description. The inventors expect skilled artisans to employsuch variations as appropriate, and the inventors intend for theinvention to be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contradicted by context.

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What is claimed is:
 1. A plant promoter selected from the groupconsisting of: (a) a plant promoter comprising the nucleotide sequenceset forth in SEQ ID NO:2; (b) a plant promoter consisting of thenucleotide sequence set forth in SEQ ID NO:2; (c) a plant promoterconsisting of the nucleotide sequence set forth in SEQ ID NO:10; and (d)a plant promoter comprising nucleotides 2208-2456 set forth in SEQ IDNO:10 and from 1-2207 contiguous nucleotides set forth in SEQ ID NO:10that are 5′ to and contiguous with nucleotide
 2208. 2. The plantpromoter of claim 1, wherein the promoter comprises the nucleotidesequence set forth in SEQ ID NO:2.
 3. The plant promoter of claim 1,wherein the promoter consists of the nucleotide sequence set forth inSEQ ID NO:2.
 4. The plant promoter of claim 1, wherein the promoterconsists of the nucleotide sequence set forth in SEQ ID NO:10.
 5. Theplant promoter of claim 1, wherein the promoter consists of nucleotides2208-2456 set forth in SEQ ID NO:10 and from 1-2207 contiguousnucleotides set forth in SEQ ID NO:10 that are 5′ to and contiguous withnucleotide
 2208. 6. A nucleic acid molecule encoding broad-spectrumdisease resistance to bacterial blight; wherein said nucleic acidmolecule comprises the plant promoter of claim 1 operably linked to anucleotide sequence encoding a Xa10 promoter comprising the amino acidsequence set forth in SEQ ID NO:14; wherein the causative agent of thebacterial blight is Xanthomonas oryzae pv. Oryzae.
 7. The nucleic acidmolecule of claim 6, wherein the nucleotide sequence encoding the Xa10protein comprises the nucleotide sequence set forth in SEQ ID NO:13. 8.The nucleic acid molecule of claim 6, wherein the nucleotide sequenceencoding the Xa10 protein consists of the nucleotide sequence set forthin SEQ ID NO:15.
 9. The nucleic acid molecule of claim 6, wherein thenucleotide sequence encoding the Xa10 protein consists of the nucleotidesequence set forth in SEQ ID NO:16.
 10. The nucleic acid molecule ofclaim 6, wherein the nucleotide sequence encoding the Xa10 proteincomprises nucleotides 1-381 of the nucleotide sequence set forth in SEQID NO:17 and from 1-1834 contiguous nucleotides set forth in SEQ IDNO:17 that are 3′ to and contiguous with nucleotide
 381. 11. The nucleicacid molecule of claim 6, wherein the nucleotide sequence encoding theXa10 protein comprises nucleotides 1-759 of the nucleotide sequence setforth in SEQ ID NO:17 and from 1-1456 contiguous nucleotides set forthin SEQ ID NO:17 that are 3′ to and contiguous with nucleotide
 759. 12.The nucleic acid molecule of claim 7, wherein the nucleotide sequenceencoding the Xa10 protein consists of the nucleotide sequence set forthin SEQ ID NO:17.
 13. A vector comprising the nucleic acid molecule ofclaim
 6. 14. A transgenic plant cell comprising the nucleic acidmolecule of claim 6 stably integrated in its genome.
 15. A transgenicplant comprising the nucleic acid molecule of claim 6 stably integratedin its genome.
 16. The plant cell or transgenic plant of claim 14 or 15,wherein the plant is rice.
 17. The transgenic plant of claim 15, whereinthe transgenic plant has broad-spectrum disease resistance to bacterialblight; wherein the causative agent of the bacterial blight isXanthomonas oryzae pv. Oryzae.
 18. Progeny of the transgenic plant ofclaim 15, wherein the progeny is produced by selfing said transgenicplant or breeding a second plant with said transgenic plant andrecovering the progeny of the selfing or the breeding; wherein theprogeny comprise the nucleic acid.
 19. A method of preparing atransgenic plant having broad-spectrum resistance to Xanthomonas oryzaepv. Oryzae—caused bacterial blight; said method comprising introducingthe nucleic acid molecule of claim 6 into a plant, wherein thetransgenic plant has the nucleic acid molecule stably integrated in itsgenome.
 20. A method of preparing a transgenic plant havingbroad-spectrum resistance to Xanthomonas oryzae pv. Oryzae—causedbacterial blight; said method comprising transfecting the nucleic acidmolecule of claim 6 into a plant cell or plant cells and regenerating atransgenic plant from the transfected plant cell or transfected plantcells, wherein the transgenic plant has the nucleic acid molecule stablyintegrated in its genome.
 21. The method of claim 19 or 20, wherein theplant is rice.
 22. A method of preparing a transgenic plant havingbroad-spectrum resistance to Xanthomonas oryzae pv. Oryzae—causedbacterial blight; said method comprising introducing the vector of claim13 into a plant, wherein the transgenic plant has the nucleic acidmolecule stably integrated in its genome.
 23. A method of preparing atransgenic plant having broad-spectrum resistance to Xanthomonas oryzaepv. Oryzae—caused bacterial blight; said method comprising transfectingthe vector of claim 13 into a plant cell or plant cells and regeneratinga transgenic plant from the transfected plant cell or transfected plantcells, wherein the transgenic plant has the nucleic acid molecule stablyintegrated in its genome.